This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, in these and in other regards, the invention relates to novel polynucleotides and polypeptides of the ATPase family, hereinafter referred to as xe2x80x9cFtsHxe2x80x9d.
It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
The frequency of Staphylococcus aureus infections has risen dramatically in the past 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
FtsH, an essential membrane bound protein involved in membrane functions, cell cycle control and gene expression was initially characterised in Escherichia coli (Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1344-1351). The Escherichia coli FtsH protein comprises 644 amino acid residues with a predicted molecular mass of 70.7 kDa. and has been shown to localise to the cytoplasmic membrane via two hydrophobic domains (Tomoyasu, T., Yamanaka, K., Murata, K., Suzaki, T., Bouloc, P., Kato, A., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1352-1357). It belongs to a novel putative ATPase family known as the AAA-protein family, members of which are widely distributed among eubacteria, archaebacteria and eukaryotes (Kunau, W.H., Beyer, A., Franken, T., Gotte, K., Marzioch, M., Saidowski, J., Skaletz-Rorowski, A. and Wiebel, F. F. (1993) Biochemie 75: 209-224). FtsH demonstrates significant homology to these ATPases over a cytoplasmic region of some 200 amino acid residues which includes a putative ATP binding site, a zinc-binding motif and the adjacent C-terminal sequence. Recently, Escherichia coli FtsH was shown to catalyse the ATP dependent degradation of the ("sgr"32 subunit of Escherichia coli RNA polymerase (Tomoyasu, T., Gamer, J., Bukau, B., Kanemori, M., Mori, H., Rutman, A. J., Oppenheim, A. B., Yura, T., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1995) The EMBO Journal 14 2551-2560) and as such is thought to be a key element in transcriptional control. In addition, FtsH is required for the proteolytic elimination of uncomplexed forms of SecY, important in maintaining optimal protein translocation and integrity of the membrane (Kihara, A., Akiyama, Y., Ito, K. (1995) Proceedings of the National Academy of Sciences USA 92: 4532-4536). Overproduction of SecY in FtsH mutant cells has been shown to deleteriously effect cell growth and protein export.
In addition to Escherichia coli (Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1344-1351), highly conserved FtsH homologues have been identified in Lactococcus lactis (Nilsson, D., Lauridsen, A. A., Tomoyasu, T. and Ogura, T. (1994) Microbiology 140: 2601-2610), Bacillus subtilis, (Ogasawara, N., Nakai, S. and Yoshikawa, H. (1994) DNA Research 1: 1-14), and Saccharomyces cerevisiae (Thorsness, P. E., White, K. H. and Fox, T. D. (1993) Molecular and cellular Biology 13: 5418-5426, Schnall, R., Mannhaupt, G., Stuka, R., Ehnle, S., Schwarzlose, C., Vetter, I. and Feldmann, H. (1994) Yeast 10 1141-1155) however with the exception of Escherichia coli FtsH, none of these proteins have been purified and studied biochemically. The high level of identity among diverse eubacteria and eukaryotes strongly suggests commonality of function. The ftsH gene is essential for cell viability in Escherichia coli (Tomoyasu, T., Yuki, T., Morimura, S., Mori, H., Yamanaka, K., Niki, H., Hiraga, S. and Ogura, T. (1993) Journal of Bacteriology 175: 1344-1351). Inhibitors of FtsH proteins would prevent bacteria from establishing and maintaining infection of the host by disrupting transcription and protein translocation, resulting in arrested growth and ultimately to cell death as the bacteria become susceptible to host defences and thereby have utility in anti-bacterial therapy.
Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
The polypeptides of the invention have amino acid sequence homology to a known Bacillus subtilis FtsH protein.
It is an object of the invention to provide polypeptides that have been identified as novel FtsH polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO:2] and a known amino acid sequence or sequences of other proteins such as Bacillus subtilis FtsH protein.
It is a further object of the invention to provide polynucleotides that encode FtsH polypeptides, particularly polynucleotides that encode the polypeptide herein designated FtsH.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding FtsH polypeptides comprising the sequence set out in Table 1[SEQ ID NO:1], or a variant thereof.
In another particularly preferred embodiment of the invention there is a novel FtsH protein from Staphylococcus aureus comprising the amino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.
In accordance with another aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain contained in NCIMB Deposit No. 40771.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding FtsH, particularly Staphylococcus aureus FtsH, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of FtsH and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of Staphylococcus aureus referred to herein as FtsH as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
Among the particularly preferred embodiments of the invention are variants of FtsH polypeptide encoded by naturally occurring alleles of the FtsH gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned FtsH polypeptides.
In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing FtsH expression, treating disease, for example, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis), assaying genetic variation, and administering a FtsH polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Staphylococcus aureus bacteria.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to FtsH polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention there are provided antibodies against FtsH polypeptides.
In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypetide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
In accordance with yet another aspect of the invention, there are provided FtsH agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists.
In a further aspect of the invention there are provided compositions comprising a FtsH polynucleotide or a FtsH polypeptide for administration to a cell or to a multicellular organism.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
xe2x80x9cHost cellxe2x80x9d is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
xe2x80x9cIdentity,xe2x80x9d as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, xe2x80x9cidentityxe2x80x9d also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. xe2x80x9cIdentityxe2x80x9d and xe2x80x9csimilarityxe2x80x9d can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
xe2x80x9cIsolatedxe2x80x9d means altered xe2x80x9cby the hand of manxe2x80x9d from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not xe2x80x9cisolated,xe2x80x9d but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is xe2x80x9cisolatedxe2x80x9d, as the term is employed herein.
xe2x80x9cPolynucleotide(s)xe2x80x9d generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. xe2x80x9cPolynucleotide(s)xe2x80x9d include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, xe2x80x9cpolynucleotidexe2x80x9d as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the termn xe2x80x9cpolynucleotide(s)xe2x80x9d also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are xe2x80x9cpolynucleotide(s)xe2x80x9d as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term xe2x80x9cpolynucleotide(s)xe2x80x9d as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. xe2x80x9cPolynucleotide(s)xe2x80x9d also embraces short polynucleotides often referred to as oligonucleotide(s).
xe2x80x9cPolypeptide(s)xe2x80x9d refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. xe2x80x9cPolypeptide(s)xe2x80x9d refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. xe2x80x9cPolypeptide(s)xe2x80x9d include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formnation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-arboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINSxe2x80x94STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
xe2x80x9cVariant(s)xe2x80x9d as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid due may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.